The goal of these proposed studies is to understand the molecular mechanism of homologous genetic recombination in mammalian cells. Homologous recombination plays an integral role in maintaining genomic stability and is an important mechanism for the repair of chromosomal double-strand breaks resulting from exposure to ionizing radiation. Our work focuses on the functional organization of domains within the human Rad52 protein (HsRad52) as well as the functional relationship(s) between HsRad52 and other human recombination proteins. Despite significant similarities in the overall mechanism of DNA strand exchange in bacteria, yeast and higher eukaryotes, the larger number of components involved in the regulation and catalysis of recombination in vertebrates suggests a considerably more complex process. We have identified a new self-association domain and a potential second DNA binding site within HsRad52. Our studies will address the functional relevance of these and other protein domains both in vitro and in vivo. Genetic studies will be performed using chicken B cell lines in which mutant RAD alleles are complemented by their human counterpart. We will also investigate the use of RNAi for our genetic studies. This combination of methods will provide important insights into specific biochemical aspects of HsRad52 that are required for optimal function in vivo. Understanding the molecular mechanistic principles of homologous recombination in humans has far-reaching effects for creating novel proteins with desired properties that may be used for prevention of diseases resulting from genomic instability and for beneficial genetic manipulation.